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Impact of Propionic Acid on Liver Damage in Rats
Sooad Al- Daihan and Ramesa Shafi Bhat∗
Biochemistry Department, Science College, King Saud University, Riyadh, Saudi Arabia.
Propionic acid (PA) is a short chain fatty acid, a common food preservative and metabolic end product of enteric
bacteria in the gut. The present study was undertaken to investigate the effect of PA on liver injury in male rats.
Male western albino rats were divided into two groups. The first group served as normal control, the second was
treated with PA. The activities of serum hepatospecific markers such as aspartate transaminase, alanine
transaminase, and alkaline phosphatase were estimated. Antioxidant status in liver tissues was estimated by
determining the level of lipid peroxidation and activities of enzymatic and non-enzymatic antioxidants. Sodium
and potassium levels were also measured in liver tissue. PA treatment caused significant changes in all
hepatospecific markers. Biochemical analysis of liver homogenates from PA-treated rats showed an increase in
oxidative stress markers like lipid peroxidation and lactate dehydrogenase, coupled with a decrease in
glutathione, vitamin C and glutathione S- transferase. However, PA exposure caused no change in sodium and
potassium levels in liver tissue. Our study demonstrated that PA persuade hepatic damage in rats.
Key words: Propionic acid, liver, oxidative stress
∗
Corresponding author: Biochemistry Department, Science College, King Saud University, P,O box 22452, Zip code 11495, Riyadh, Saudi Arabia. E-mail: [email protected], [email protected]
ropionic acid (PA) is a naturally occurring
carboxylic acid. This dietary short-chain fatty
acid occurs naturally in milk, dairy products such as
yogurt and cheese (1, 2). PA is mainly produced by
the fermentation of indigested food by the
microbiota in the colon, but can reach the blood
compartment and the adipose tissue, where it
reduces fatty acid levels in plasma via the inhibition
of lipolysis and induction of lipogenesis in adipose
tissue and suppression of fatty acid production in
liver (3). PA is a fungicide and bactericide and is
therefore used as a food preservative (4, 5). It is
also used to control fungi and bacteria growth in
stored grains, hay, grain storage areas, poultry litter
and drinking water (6). Pharmaceutical companies
commonly include PA in the formulation of
steroidal and nonsteroidal anti-inflammatory
medi-cations. Fluticasone inhalers used for respiratory
conditions, antihistamine and decongestant,
comm-only contain PA.
PA derived from colonic bacterial
fermen-tation contributes substantially to overall propionate
load in children with disorders of propionate
metabolism.The gut microbiota and its metabolites
such as PA can access the brain through various
routes within the liver – gut– brain axis (7, 8).The
gut and the liver are intimately associated, and there
is continuous bidirectional communication between
P
Submmited 3 February 2015; Accepted 14 March 2015; Published 28 June 2015
these organs through the bile, hormones,
inflammatory mediators, and the products of
digestion and absorption. In the colon, PA is the
metabolic end product of enteric bacteria, produced
by fermentation of polysaccharides,
oligosaccha-rides, long- chain fatty acids, proteins, peptides and
glycoprotein precursors (9). The majority of the PA
produced in the colon is absorbed, passes the
colonocytes and the viscera, and drains into the
portal vein. Around 90% of PA is metabolized by
the liver and the rest is transported into the
peripheral blood (10). Propionate affects various
metabolic processes such as gluconeogenesis,
ureogenesis or ketogenesis (11). However, there are
also findings which appear inconsistent with the
physiological role of propionate in the control of
carbohydrate or lipid synthesis (12). PA is mainly
metabolized in the liver and has been shown to
inhibit gluconeogenis and increases glycolysis in rat
hepatocytes (13). It has also been proposed that
propionic acid may lower plasma cholesterol
concentrations by inhibiting hepatic
cholestero-genesis (14). PA influences the production
of hormones through adipose tissue, such as
the induction of leptin, which is a potent
anorexi-genic hormone and suppresses food intake through
receptors expressed in the central nervous
system (15).
Even though PA is necessary for normal
immune and physiological functioning; elevated
levels may result in disruptive effects.(16). PA can
readily cross the gut- liver- blood- brain barriers
and gain access to the central nervous system (17).
In the brain, it can cross cell membranes and
accumulate within cells, inducing intracellular
acidification (18, 19) which may alter
neurotrans-mitter releases and, ultimately, neuronal
communi-cation and behavior (20, 21). In fact, propionic
acidemia is a neurodevelopmental metabolic
disorder characterized by elevated levels of PA that
clinically resembles some aspects of autism (22).
Liver is among the most important organs
involved in metabolic activities. The intracellular
enzymes of the parenchyma of liver cells have an
amazing capacity to perform diverse and
complicated biochemical functions. An elevation in
the levels of serum marker enzymes with oxidative
stress is generally regarded as one of the most
sensitive indexes of hepatic damage (23). PA is
neurotoxic and its axis to brain is through liver (7,
8).This study attempted to investigate the possible
role of neurotoxic dose of PA on liver injury in
male rats as the majority of PA is metabolized by
the liver.
Materials and methods
Animals
Adult male western albino rats weighing
150-200 g purchased from the animal house of science
college, King Saud University, Riyadh were used
throughout this study. The animals were fed on
standard pellet diet and water ad libitum. The
animals were maintained in a controlled
environ-ment under standard conditions of temperature and
humidity with an alternating light- and– dark cycle.
All the procedures described were reviewed and
approved by the King Saud University Animal
Ethics Committee.
Dosage and treatment
Rats were randomly divided into two groups
with ten rats in each. The first group served as a
control. On the eighth day, the animals of the
second group given an oral dosage of PA at the
dose of (250 mg/kg body weight/day for three days;
n= ten) (24). On the third day of PA administration,
the rats were scarified and the liver organ was
isolated.
Sample preparation
Blood collection for estimation of AST, ALT and
ALP
The blood was collected from retro-orbital
plexus without the use of anticoagulant. The blood
was allowed to stand for 10 min before being
centrifuged at 2000 rpm for 10 min to obtain serum
for analysis of alanine aminotransferase (ALT),
aspartate aminotransferase (AST) and alkaline
phosphatase (ALP)
Tissue preparation
Liver tissue was washed in ice cold normal
saline and homogenized in a homogenizing buffer
(50 mM Tris- HCl, 1.15% KCl pH 7.4) using
Teflon homogenizer. The homogenate was
centrifuged at 4000 g for 20 minutes to remove
debris and kept at−80 °C until further use. The
supernatant was used for the estimation of
glutathione, lipid oxidation,
glutathione-S-transferase, lactate dehydrogenase, potassium,
sodium and vitamin C.
Biochemical analysis
Serum alanine aminotransferase (ALT)
ALT was estimated by the method of Reitman
and Frankel (25). Briefly, 0.5 ml of substrate (2
mM α-ketoglutarate, 0.2 M DL-alanine in 0.1M
phosphate buffer pH 7.4) was incubated at 37 oC for 5 minutes. 0.1 ml of freshly prepared serum was
added to the aliquot and again incubated at 37 oC for 30 minutes. At the end of incubation, 0.5 ml of
2, 4-dinitrophenyl hydrazine was added, and the
aliquot left for 30 minutes at room temperature. 0.5
ml of 0.4 N NaOH was added, and the aliquot was
again left for 30 minutes. Absorbance was then
recorded at 505 nm against water blank.
Serum aspartate aminotransferase (AST)
AST was estimated by the method of Reitman
and Frankel (25). The substrate, however, was 2
mM α-ketoglutarate, 0.2 M DL-aspartate, and the
rest of the procedure was similar to ALT
measurement method.
Serum alkaline phosphatase (ALP)
Serum alkaline phosphatase activity was
measured according to the method of King and
Armstrong (26), using disodium phenyl phosphate
as substrate. The colour developed was read at
510 nm.
Measurement of lipid peroxidation
Lipid oxidation was evaluated by measuring the
levels of lipid peroxidation by-products as
thiobarbituric acid reactive substances (TBARS),
namely malondialdehyde (MD), using the method
of Ruiz-Larrea et al. (27). Accordingly, the samples
were heated with TBA at low pH and the formation
of a pink chromogen was measured by absorbance
at 532 nm. The concentration of lipid peroxides was
calculated as µ moles/ml using the extinction
coefficient of MD.
Assay of vitamin C
Assay of vitamin C was performed according to the
method of Jagota and Dani (28). 0.2 ml of liver
homogenate was mixed with 0.8 ml of 10%
trichloroacetic acid (TCA) and incubated in ice for
5 minutes. The samples were then centrifuged for
10 minutes at 3500 rpm and 4°C. 1.5 ml double
distilled water was subsequently added to 0.5 ml of
the supernatant. Eventually, 2 ml of Folin-phenol
reagent was added and absorbance was measured at
760 nm after 10 minutes.
Assay of glutathione (GSH)
GSH content was determined according to the
method described by Beutler et al. (29) using 5,5′
-dithiobis 2-nitrobenzoic acid (DTNB) with
sulfhy-dryl compounds to produce a relatively stable
yellow color.
Glutathione-S-transferase (GST) assay
The GST activity was determined by the
method of Habig et al. (30). The
1-chloro-2-4-di-nitrobenzene (CDNB) is neutralized by the enzyme
in the presence of GSH as a cosubstrate. The
change in absorbance is measured at 340 nm and
the activity is expressed as nmol/min/mg protein.
Assay of lactate dehydrogenase (LDH)
The quantitative determination of LDH in the
brain homogenates was performed using the
lactate-to-pyruvate kinetic method described by Henry et
al. (31).
Determination of potassium levels
Potassium levels were measured in a protein-
free alkaline medium by reaction with sodium
tetraphenyl boron, which produced a colloidal
suspension. The turbidity of such a suspension is
proportional to the potassium concentrations in the
range of 2–7 mmol/ l (32).
Determination of sodium levels
Sodium levels were assayed by enzymatic
determination of sodium, i.e., the measurement of
sodium-dependent galactosidase activity using
ortho-Nitrophenyl- β- galactoside (ONPG) as a substrate (33)
Statistical analysis
The values are expressed as mean ± standard
error of the mean (SEM). The results were
evaluated using the SPSS (version 12.0) and Origin
6 softwares and evaluated by One -way ANOVA
complemented with the Dunnett’s test for multiple
comparisons. Statistical significance was
considered when The p-value was < 0.05
Results
Effect of PA on hepatic markers
The activities of AST, ALT and ALP are
presented in Table 1. We observed statistically a
significant increase in AST and ALP in serum of
rats exposed to PA (250 mg/kg body weight/day for
three days). Also the administration of PA induced
a marked increase in ALT levels as compared to the
control group.
Effect of PA on GSH and antioxidant enzyme
activities
Table 2 presents the mean ± SEM of the GSH
(µg/ml), MD (µmoles/ml) and vitamin C (µg/ml)
concentrations, GST (U/ml) and LDH activities in
the liver homogenates of the two groups of rats.
Compared to control groups, the PA-treated rats
exhibited a statistically significant increase in MD
and LDH activities with a concomitant decrease of
GST, GSH, and Vitamin C.
Table 1. Serum AST, ALT and ALP activities
Parameters Groups Min Max Mean ± SEM P value
AST (U/L) Control 139.67 154.7 147.62±3.38 0.01
PA 203.32 365.09 247.34±30.09
ALT (U/L) Control 52.45 104.31 84.27±9.17 0.09
PA 98.41 174.44 117.03±14.43
ALP (U/L) Control 365.7 615.48 496.24±43.24 0.05
PA 533.37 862.5 664.884±61.31
AST: serum aspartate aminotransferase; ALT: serum alanine aminotransferase; ALP: serum alkaline phosphatase;.max: maximum; min: minimum; PA: propionic acid; SEM: standard error of the mean.
Table 2. GSH, MD, vitamin C concentrations and GST and LDH activities in the liver homogenates
Parameters Groups Min Max Mean± SEM P value
GSH(µg /ml) Control 57.97 79.71 66.18±4.62 0.01
PA 24.15 50.72 45.40±5.31
MD (µmoles /ml) Control 0.113 0.151 0.13±0.007 0.01
PA 0.16 0.33 0.23±0.030
Vitamin C (µg /ml) Control 39.4 100 68.75±11.09 0.04
PA 37.8 39.4 38.48±0.32
GST (U /ml) Control 38.75 54.27 48.24±3.05 0.05
PA 3.08 40.83 30.54±7.09
LDH (U /L) Control 130.43 161.83 148.77±5.43 0.001
PA 185.99 239.13 208.2±10.78
GSH: glutathione; MD: malondialdehyde; LDH: lactate dehydrogenase; max: maximum; min: minimum; SEM: standard error of the mean.
Fig. 1. Percentage change of all parameters in PA group compared to control.
Table 3. Sodium and potassium levels in the liver homogenates.
Parameters Groups Min Max Mean ± SEM P value
Potassium mmol/L
Control 7.6 8.48 8.05±0.69 0.06
PA 8.22 8.93 8.52±0.45
Sodium mmol/L
Control 114.3 118.10 116.53±0.16 0.62
PA 115.1 117.3 116.94±0.12
max: maximum; min: minimum; SEM: standard error of the mean
Effect of PA on sodium and potassium
In liver tissue, sodium and potassium levels
were not affected by PA treatment as shown in
Table 3. Figure 1 shows the percentage change of
all parameters in PA treated group compared to
control.
Discussion
The levels of some important biochemical
parameters in serum are used as diagnostic markers
of hepatic injury. One of the most sensitive and
dramatic indicators of hepatocyte injury is the
release of intracellular enzymes, such as
transaminases and serum alkaline phosphatase. The
elevated activities of these enzymes are indicative
of cellular leakage and the loss of the functional
integrity of the cell membranes in liver which are
always associated with hepatonecrosis (34-36). The
oral administration of PA showed drastic alterations
in the level of serum marker enzymes AST, ALT
and ALP when compared with control rats as
shown in Table 1, indicating PA mediated hepatic
damages. ALP is a membrane associated enzyme
and an increased activity of ALP is an indication of
liver damage (37). ALT is more abundant in the
liver cells than in any other cells in the body and is
primarily used as a specific marker of hepatic
damage. ALT and AST enzymes are regarded as
markers of liver injury since liver is the major site
of metabolism (38, 39).
Liver plays a pivotal role in the regulation of
various physiological processes in the body such as
carbohydrate metabolism and storage, fat
metaboli-sm, bile acid synthesis, and so forth besides being
the most important organ involved in the
detoxification of various drugs as well as
xenobiotics in our body (40). Antioxidant enzymes
act in a coordinated fashion to prevent the oxidative
stress. Thiobarbituric acid reactive substances
(TBARS), the final metabolites of peroxidized 0
20 40 60 80 100 120 140 160 180 200
%
c
h
a
n
g
e
control
PA
polyunsaturated fatty acids and the late biomarker
of oxidative stress (41) were significantly increased
in our results, providing a direct assessment of the
progression of liver injury at the cellular level
(42).The increased level of LDH in PA treated
group is an indication of abnormality in liver
functioning, which may be due to the formation of
highly reactive free radicals due to PA treatment.
Reduced glutathione (GSH) is an important
reductant in the cell, where it protects against free
radicals, peroxides and other toxic components. It
maintains the normal structure and function of cells,
probably by its redox and detoxification reactions
(43). The decreased concentration of GSH in liver
tissue found in PA treated group of our study may
be due to NADPH reduction or GSH utilization in
the exclusion of peroxides (44). Support for this
comes from some previous findings (21, 45) in
which the high levels of PA were reported to induce
oxidative stress with decreased levels of total GSH
in brain tissue.
Circulating antioxidants such as vitamin C are
non-enzymatic scavengers of free radicals. A
decrease in ascorbic acid levels in plasma (46) and
liver (47) was reported in injured liver in rats.
Decreased levels of vitamin C found in PA treated
rats in the present study are in line with the findings
of a previous study by El-Ansary et al. (45).
Glutathione-S-transferase is actually
compos-ed of a group of isoenzymes capable of detoxifying
various exogenous and endogenous substances by
conjugation with glutathione. A reduction in the
activity of these enzymes is associated with the
accumulation of highly reactive free radicals,
leading to deleterious effects such as loss of
integrity and function of cell membranes (48, 49).
Significant decrease of GST reported in the present
study could easily be related to the oxidative effect
of PA previously reported by Alfawaz et al. (50).
The active transport of sodium- potassium
across the cell membrane is controlled by sodium-
potassium-adenosine triphosphatase
(Na+-K+-ATPase) enzyme, which is an integral plasma
membrane protein responsible for a large part of the
energy consumption constituting the cellular
metabolic rate. Na+-K+-ATPase controls cell
volume, nerve and muscle signals and drives the
transport of amino acids and sugars (51). However,
the non significant difference from the control value
by administration of PA suggests that it may be of
low toxicity to the monovalent ion.
In conclusion, increased activities of AST,
ALT, and ALP suggest severe hepatic injury
resulting from the administration of PA. The
increase of TBARS in liver of treated rats provides
evidence for the pathogenic role of PA in inducing
oxidative liver injury.
Acknowledgment
This research project was supported by a grant
from the “Research Center of the Center for Female
Scientific and Medical Colleges”, Deanship of
Scientific Research, King Saud University.
Conflict of interests
The authors declared no Conflict of interests.
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